1) Huanglongbing (HLB) is a disease affecting citrus trees that is caused by a bacterium and spread by an insect. It reduces fruit yield and quality and ultimately kills trees. 2) HLB symptoms can take years to appear, allowing the disease to spread widely before detection. Earlier detection is needed to prevent further spread and losses. 3) Researchers are using metabolomics to detect early physiological changes in trees infected with the HLB bacterium, before visual symptoms appear. They hope to identify reliable metabolic biomarkers for improved early detection of the disease.

1. 32 Citrograph Vol. 6, No. 2 | Spring 2015
crb-funded Research Progress report
Early Detection of HLB
with Metabolomics
Elizabeth Chin, Rebecca Lobo, John da Graça, Mark Hilf, Greg McCollum,
Cynthia LeVesque, Kris Godfrey and Carolyn Slupsky
Huanglongbing (HLB) is a major threat to the global citrus
industry. The name literally translates to‘Yellow Shoot Disease,’
emphasizing the yellow mottle of symptomatic leaves and its
devastatingimpactoncitrustrees. Infectedtreeshavereducedfruit
yieldduetoprematurefruitdropandpoorfruitqualitycomparedto
uninfected trees. Symptomatic fruit from infected trees are small in
size, lopsided in shape and green in color. Even apparently“normal”
looking fruit from infected trees may experience changes in flavor,
often acquiring a bitter and/or metallic taste that is unpalatable to
consumers. Ultimately, diseased trees die prematurely compared to
uninfected trees.
Unfortunately, the symptoms of HLB can take years to appear and
resembleothercitrusdiseasesandnutrientdeficiencies.Meanwhile,
the Asian citrus psyllid (ACP), the insect vector associated with the
disease, can still transmit the bacterium from infected trees to
healthy trees even when the infected trees are asymptomatic. The
long period between infection and the appearance of symptoms
allows the disease to spread widely and can result in huge losses
in fruit, trees and profit. Indeed, Florida’s citrus industry has already
been devastated by HLB with billions of dollars and thousands of
jobs lost. California’s citrus industry now faces the same outcome
unless disease spread can be prevented by earlier detection of
infection in non-symptomatic trees.
HLB-infected tree displaying fruit drop.

2. www.CitrusResearch.org | Citrograph Magazine 33
In the U.S., HLB is caused by the bacterium“Candidatus Liberibacter
asiaticus” (CLas) and is spread by the insect known as the Asian
citrus psyllid (ACP), or by grafting infected tissue. Resistance has not
been identified in commercial citrus varieties; and if infected with
CLas, these trees will eventually succumb to the disease. Research
on CLas is especially challenging since the bacterium has yet to
be grown in the laboratory, so there is no way to study it outside
of the plant. Most efforts to reduce the spread of CLas are aimed
at controlling ACP populations by trapping, use of bio-parasites
or spraying insecticides. However, there are currently no fail-safe,
long-term solutions for treating the disease or controlling spread in
field trees other than tree removal.
Currently, the only method with regulatory approval for CLas
detection is quantitative polymerase chain reaction (qPCR), which
provides a measure of the amount of CLas based on detecting DNA
from the pathogen. Unfortunately, CLas is not evenly distributed
throughout the tree. Since the accuracy of qPCR depends on
whether the sample contains the CLas bacterium, qPCR can yield
false-negative results if the correct sample is not assayed. This is
especially problematic in truly infected trees that are asymptomatic
and, therefore, resemble healthy trees because there are no visual
symptoms to guide leaf sampling. Thus, the inability to detect the
pathogen during asymptomatic infection means that many trees
are clandestinely carrying the disease and allowing others to be
infected.
Although the bacteria may be present at low levels within the tree
and difficult to detect by qPCR, the tree can detect the presence
of the pathogen and mount a physiological response. It has been
well documented that plants can defend themselves against
stressors such as changes in temperature or water availability,
as well as against insect feeding and pathogens. Initiation of the
plant’s defense system leads to a cascade of changes in metabolic
pathways to create signal molecules, enzymes, proteins and other
biochemical machinery. These changes occur soon after infection,
and measuring the changes in quantity of the participating
metabolites can identify the metabolic pathways that shift in the
plant.
Measuring the multitude of metabolites in a sample is a daunting
task, as there can be hundreds or thousands of these chemicals
that are present in concentrations ranging from very low to very
high. No one technique can measure all of the metabolites present
in a leaf, but one of the most robust and reproducible analytical
instruments for measuring metabolites that range in concentration
by a factor of more than one million is the Nuclear Magnetic
Resonance (NMR) instrument (Figure 1). NMR works by placing
a sample into a magnetic field and applying a radio-frequency
pulse. The resulting oscillating electrical signals can be detected in
a receiver coil. These signals are then processed into a frequency
spectrum that can be matched to a library of unique metabolite
signatures, allowing for the identification of metabolites present
Figure 1. Analysis of leaf samples by NMR. Leaf
samples are extracted and placed in NMR tubes
for data collection on the NMR spectrometer.
The resulting spectra are analyzed for metabolite
content.

3. 34 Citrograph Vol. 6, No. 2 | Spring 2015
in a sample. Measurement of the area under the spectrum allows
for quantitation. Understanding the CLas-induced ‘metabolite
fingerprint,’ i.e. which metabolites change and by how much, can
potentially be used as an improved early detection method for HLB
in citrus trees.
Research in our lab has consistently shown that there is a CLas-
specific metabolite fingerprint that may be used for the detection
of CLas earlier than qPCR, and prior to visual symptoms. Early
detection of HLB allows for early intervention that will help prevent
the spread of the disease to other trees, thus reducing losses in
trees, fruit and profit to the citrus industry. Greenhouse trees as
well as field trees from California and Texas are being studied to
gain a comprehensive overview of which metabolites change soon
after CLas infection, how they continue to change during disease
progression, and how this knowledge can be applied to detect CLas
in mature, fruit-bearing trees soon after infection (Figure 2).
GreenhouseTreesSince the CLas bacterium cannot be grown in the laboratory, we
have been studying plants grown in greenhouses in both Florida,
and at the University of California-Davis Contained Research Facility
(for more about all HLB early detection groups in the CRF, please
see “An Interdisciplinary Approach to Combat HLB” in Citrograph,
Winter 2014).
We are investigating which metabolites change over the course of
infection in several varieties of citrus to establish the earliest time at
which metabolomics can detect infection. Because we are able to
see changes in the metabolite patterns, these studies may enable
us to determine the length of time that a tree has been infected,
which will aid in monitoring the spread and help determine how
long an infection has been in an area. We also have been studying
theeffectsofothercitruspathogens,suchasCTV,Xanthomonascitri
subspecies citri (bacterial citrus canker) and Spiroplasma citri (citrus
stubborn), and have determined that their metabolite profiles are
distinct from CLas. These studies are building the foundation for
our metabolite-based early detection method.
FieldTreesIn tandem with the CRF greenhouse experiment, we are validating
our test using field samples collected from a grapefruit orchard in
Texas where HLB has been confirmed by qPCR in a small number
of trees with foliar symptoms on a few branches. The samples are
being collected from neighboring and nearby trees that are most
likely to be infected next. Some leaves are used for qPCR analysis in
the da Graça lab while the remainder are lyophilized and sent to the
Slupsky lab for metabolomics analysis. The results from this study
will help us refine and validate our biomarker profiles to ensure that
very few false positives and false negatives are obtained using our
detection method.
Improvements for detecting CLas infection are essential to
combatting the spread of HLB. Early detection of HLB allows for
earlierintervention(treeremovaland,perhapseventually,treatment
of the diseased tree), which will play a key role in preserving the
citrus industry. Metabolomics offers a promising new strategy for
the early detection of, defense against and resolution of HLB in the
United States and the survival of the citrus industry.
Elizabeth Chin is a graduate student in the Department of Food
Science and Technology at the University of California, Davis;
Rebecca Lobo, Ph.D., is a post-doctoral fellow in the Department
of Food Science and Technology at the University of California,
Davis; JohndaGraça,Ph.D.,isaprofessoratTexasA&MUniversity
in Weslaco Texas; Mark Hilf, Ph.D., is a research plant pathologist
at the U.S. Horticultural Research Laboratory at the USDA in
Florida; Greg McCollum, Ph.D., is a research plant physiologist at
theU.S.HorticulturalResearchLaboratoryattheUSDAinFlorida;
Cynthia LeVesque, Ph.D., is the laboratory director of the Citrus
Research Board in Riverside, California; Kris Godfrey, Ph.D., is
an associate project scientist at the Contained Research Facility
at the University of California, Davis; and Carolyn Slupsky,
Ph.D., is an associate professor with a joint appointment in the
Department of Nutrition and the Department of Food Science
and Technology at the University of California, Davis.
Figure 2. Overview of the plan to obtain a set of reliable
markers for detection of CLas infection in citrus.